CN1883465A - Apoferritin -platinum anticancer drug composition and preparation method thereof - Google Patents
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- 108010002084 Apoferritins Proteins 0.000 title claims abstract description 62
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 61
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- 229940041181 antineoplastic drug Drugs 0.000 title claims abstract description 51
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- 238000000034 method Methods 0.000 claims abstract description 35
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- 238000000502 dialysis Methods 0.000 claims abstract description 12
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- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 claims abstract description 5
- 229960004316 cisplatin Drugs 0.000 claims description 55
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- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 34
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- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 9
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 7
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Abstract
Disclosed is an anti-cancer compound medicinal preparation of apoferritin-platinum and process for preparation, which comprises subjecting ferritin to dialysis in acetic acid buffer solution with pH of 5.0-6.0, charging sodium hydrosulfite into dislysate as reducing agent and dipyridine as chelating agent, changing dislysate every 8 hours to obtain apoferritin, then coating platinum group anti-cancer drugs into apoferritin through depolymerized recombination method or in-situ generating method.
Description
Technical Field
The invention relates to a apoferritin-platinumanti-cancer drug compound for solving the problem of administration of platinum anti-cancer drugs and a preparation method thereof.
Background
Platinum-based anticancer drugs, particularly cisplatin and carboplatin, have been used to treat various cancers, and have the following structures:
cisplatin carboplatin
At present, the platinum anticancer drugs are mainly administered by intravenous drip or arterial infusion route. However, in practical application, the clinical application is greatly limited because the drugs have serious toxic and side effects, such as renal toxicity, neurotoxicity, bone marrow toxicity and the like, and frequently encounter the problem of secondary drug resistance. Targeted drug delivery is one of the effective approaches to solve these problems, and how to select a targeted drug carrier is the key to solve the problems. Because certain protein receptors on cancer cell membranes are over-expressed, the possibility of establishing protein molecules as targeting drug carriers is provided for people.
Ferritin is a globular protein composed of 24 subunits, with 8 hydrophilic channels of about 0.4nm size opening into the interior of the protein at the intersection of the subunits. The protein coat encloses an iron core, the iron of which is predominantly in the form of FeO (OH). After reducing the iron to a +2 valence with a reducing agent, the iron may form a complex with a chelating agent leaving the ferritin, thereby creating a cavity within the ferritin, a protein without iron called apoferritin. The preparation of apoferritin has been reported in the literature (see SimonettaStefanin, archives of Biochemistry and Biophysics, 1996, 325, p58-64), which is obtained by dialyzing ferritin in an acetic acid buffer solution (pH 5.5), adding sodium dithionite as a reducing agent to the dialysate, and exchanging the dialysate every 8 hours until the dialysate is colorless and the protein is light yellow. The internal diameter of the cavity of apoferritin was 8 nm. The apoferritin coat still retains the essential biochemical properties of ferritin and thus is still recognized by apoferritin receptors overexpressed on cancer cell membranes.
Disclosure of Invention
The invention aims to: aiming at the problems that the prior platinum anticancer drugs adopt the existing administration route, have large toxic and side effects and are easy to generate secondary drug resistance, the apoferritin-platinum anticancer drug compound taking apoferritin as a drug carrier and the preparation method thereof are provided.
The purpose of the invention is realized as follows: a apoferritin-platinum anticancer drug compound is characterized in that: the platinum anticancer drug is wrapped in apoferritin.
The platinum anticancer drug is cisplatin, carboplatin or a mixture of cisplatin and antiplatin.
The preparation method of the apoferritin-platinum anticancer drug compound is characterized by comprising the following steps of:
a) putting ferritin in acetic acid buffer solution with pH of 5.0-6.0 for dialysis, adding sodium hydrosulfite as a reducing agent and bipyridine as a chelating agent into dialysate, and replacing the dialysate every 8 hours until the dialysate is colorless and the protein is light yellow to obtain apoferritin;
b) the apoferritin and the platinum anticancer drugs are wrapped in the apoferritin by a depolymerization recombination method or an in-situ generation method to obtain the apoferritin-platinum anticancer drug compound.
In the preparation method of the invention, the platinum anti-cancer drug wrapped in apoferritin by the depolymerization and recombination method is cisplatin or carboplatin, and the preparation method specifically comprises the following steps: adding apoferritin into a saturated solution of a platinum anti-cancer drug, wherein the saturated solution of the platinum anti-cancer drug is a saturated solution of cisplatin or carboplatin, adjusting the pH value to 2.0-3.0 by using an acidic adjusting solution, slightly shaking for 30 minutes, adjusting the pH value to 6.0-9.0 by using an alkaline solution, and after incubation for two hours, completely dialyzing by using 0.1-0.2 mol/L sodium chloride solution to separate the apoferritin coated with the platinum anti-cancer drug from drug molecules which are not coated in the solution, thereby obtaining the apoferritin-platinum anti-cancer drug compound.
In the disaggregation recombination method: diluting the apoferritin by using a sodium chloride aqueous solution, and then adding the diluted apoferritin into a saturated solution of a platinum anti-cancer drug, wherein the addition amount of the apoferritin in the saturated solution of cisplatin or carboplatin is as follows: the molar ratio of the apoferritin to the cisplatin in the saturated solution is 1: 1500-2500, and the molar ratio of the apoferritin to the carboplatin in the saturated solution is 1: 3000-4000; the acidic adjusting solution is hydrochloric acid adjusting solution, and the alkaline adjusting solution is sodium hydroxide solution.
In the preparation method of the invention, the platinum anticancer drug generated in the apoferritin by the in-situ generation method is a mixture containing cisplatin and anti-platinum, and the preparation method comprises the following steps: adding K with the concentration 500 times that of the protein into the apoferritin solution diluted by the sodium perchlorate aqueous solution2PtCl4Slightly shaking the solution for 30 minutes, and then adding equal volume of NH with the pH value of 9.0-10.5 into the solution3-(NH4)2SO4And (3) after the buffer solution reacts for 24 hours, thoroughly dialyzing the reaction product by using a sodium perchlorate aqueous solution, and separating apoferritin coated with platinum anticancer drugs from reactants in the solution to obtain the apoferritin-platinum anticancer drug compound.
In the in situ generation process: and thoroughly dialyzing the reaction product by using 0.1-0.2 mol/L sodium perchlorate aqueous solution.
The invention has the advantages that: it uses the specific target affinity of ferritin to some cancer cells to wrap cis-platinum or carboplatin in apoferritin, so as to attain the goal of target administration. The drug delivery system can reduce the poison of the drug to normal cells, improve the treatment effect of the drug and avoid the loss of the drug in the transmission process. The apoferritin-platinum anti-cancer drug compound has the advantages of simple preparation method, easily obtained raw materials, few operation steps, good repeatability and no pollutant generation.
Drawings
FIG. 1 is a schematic representation of the structure of apoferritin-cisplatin or apoferritin-carboplatin;
FIG. 2 is a schematic diagram of the process for preparing apoferritin-cisplatin or apoferritin-carboplatin by depolymerization recombination;
FIG. 3 is a schematic diagram of the process for the preparation of apoferritin-cisplatin by in situ generation;
FIG. 4 apoferritin-15A two-dimensional nuclear magnetic resonance spectrum of the N-labeled carboplatin complex;
FIG. 5 Generation in control experiment15N-labelled cisplatin and15two-dimensional nuclear magnetic resonance spectrogram of the N-labeled anti-platinum and the hydrate thereof;
FIG. 6 is a non-denaturing electrophoretogram of apoferritin-cisplatin and apoferritin-carboplatin complexes;
FIG. 7 shows the results of experiments on the inhibition of the apoferritin-cisplatin complex on the PC-12 cell line of rat chromaffin neuroma cells.
Detailed Description
FIG. 1 discloses a model structure of apoferritin-platinum anticancer drug complex. In the figure: 1 is apoferritin and 2 is a platinum anticancer drug. As can be seen from FIG. 1, a platinum-based anticancer drug 2 is encapsulated in apoferritin 1. The preparation of apoferritin-platinum anticancer drug complex is described below with reference to the examples.
Example 1
And (3) putting 0.5mL and 85mg/mL ferritin in a dialysis bag, then putting the dialysis bag in acetic acid buffer solution with pH of 5.5 and 200mL for dialysis, adding 0.6g of sodium hydrosulfite as a reducing agent and 0.2g of bipyridyl as a chelating agent into the dialysate, and replacing the dialysate every 8 hours until the dialysate is colorless and the protein is light yellow to obtain apoferritin.
Example 2
FIG. 2 discloses the preparation process of the depolymerizing recombination method: adding the apoferritin diluted by a sodium chloride aqueous solution into a cisplatin or carboplatin saturated solution (the molar ratio can be respectively selected from 1: 1500-2500 or 1: 3000-4000), adjusting the pH value to 2.0 by using an acidic adjusting solution, slightly shaking for 30 minutes, adjusting the pH value to 7.5 by using an alkaline solution, incubating for two hours, filling the obtained solution into a dialysis bag, dialyzing for 24 hours in a sodium chloride solution (0.15mol/L NaCl), and replacing a dialyzate every 8 hours to separate the apoferritin coated with the platinum anti-cancer drug from drug molecules which are not coated in the solution, thereby obtaining the apoferritin-platinum anti-cancer drug compound. The preparation of apoferritin-cisplatin complexes by disaggregation recombination is described in detail below using cisplatin as an example.
Adding a sodium chloride aqueous solution (1.2mg, 0.015ml and 0.15mol/L NaCl) of apoferritin into a saturated aqueous solution (12mmol/L, 0.5ml and 0.15mol/L NaCl) of cisplatin, adjusting the pH value of a reaction solution to 2.0 by using a hydrochloric acid solution (0.1mol/L), standing at room temperature for 30 minutes, carefully adjusting the pH value of the solution to 7.5 by using a sodium hydroxide solution (0.1mol/L), standing at room temperature for 2 hours, filling a reaction product into a dialysis bag, dialyzing in a sodium chloride solution (0.15mol/L and 200ml) for 24 hours, and replacing the dialysate every 8 hours to obtain the apoferritin-cisplatin compound. The platinum content of the apoferritin-cisplatin compound is measured by an ICP method, the apoferritin concentration is measured by a Lowry method, and the following are calculated: each apoferritin molecule encapsulates 2 cisplatin molecules.
The apoferritin solution is added to a saturated aqueous solution of carboplatin and the apoferritin-carboplatin complex is obtained in the same manner. The platinum content of the apoferritin-carboplatin complex is measured by ICP method, the apoferritin concentration is measured by Lowry method, and the following are calculated: each apoferritin molecule encapsulates 5 carboplatin molecules.
FIG. 4 shows NMR spectra demonstrating that carboplatin is encapsulated in apo-carboplatin complexes prepared by depolymerization and recombination. In the figure: a is carboplatin encapsulated within apoferritin; b and c are shifts in small carboplatin signals encapsulated within the protein due to shielding by the protein shell.
Example 3
FIG. 3 discloses the preparation of the in situ generation method: adding K with the concentration 500 times that of the protein into the apoferritin solution diluted by the sodium perchlorate aqueous solution2PtCl4Slightly shaking the solution for 30 minutes, and then adding equal volume of NH with the pH value of 9.0-10.5 into the solution3-(NH4)2SO4Buffer solution, after 24 hours of reaction, the reaction solution is put into a dialysis bag and placed in sodium perchlorate solution for dialysis for 24 hours, the dialysis solution is replaced every 8 hours, apoferritin with cisplatin wrapped inside is separated from reactants in the solution, and apoferritin-cisplatin composite is obtainedA compound (I) is provided. The specific process is as follows:
an aqueous solution of apoferritin (8mg, 0.1ml, 0.15mol/L NaClO) was added4) And K2PtCl4Mixing the aqueous solution (7.5mg, 0.1ml) uniformly, adding NH with pH value of 9.0-10.53-(NH4)2SO4Buffer solution (0.2mol/L, 0.2ml) reacts for 24 hours at room temperature, reaction products are put into a dialysis bag and placed in sodium perchlorate solution (0.15mol/L) for dialysis for 24 hours, and the dialysate is replaced every 8 hours, so as to obtain apoferritin-cisplatin (+ anti-platinum) compound.
The related reaction formula:
the platinum content of the apoferritin-cisplatin compound is measured by an ICP method to be 60.5 mg/L; the concentration of apoferritin measured by Lowry method is 0.01 mmol/L; the control experiment proves that the Pt-NH prepared by the method3The cisplatin content is about 50%, and each apoferritin molecule is calculated to wrap 15 cisplatin molecules (and 15 anti-platinum molecules at the same time).
FIG. 5 shows the results of a control experiment15N-labelled cisplatin and15two-dimensional nuclear magnetic resonance spectrogram ([ Pt]) of N-labeled antiplatin and hydrate thereof]＝20mM，95％H2O/5％D2O, pH 5.2, 298K), in the figure: 1 is cisplatin; 2 is antiplatin; 3 is a cisplatin hydrolysate; 4 is a hydrolysis product of antiplatin. The nuclear magnetic resonance spectrum proves that: k2PtCl4(20mM) in NH3-(NH4)2SO4After reaction in a buffer solution (0.2mol/L, pH 10) at room temperature for 24 hours, [ PtCl]4]2-Cl in (1)-Ions will be partially absorbed by NH in solution3Substitution to produce a series of Pt-NH3The complex, wherein the main products are cisplatin and antiplatin. Integration of the peak areas of the relevant species in the plot gives: the ratio of the yields of cisplatin and antiplatin was 1: 1. This control experiment shows [ PtCl]access to the interior of apoferritin4]2-Under the same experimental condition, the reaction produces the mixture of cisplatin and anti-platinum, so that the apoferritin-cisplatin compound can be prepared by in-situ production method。
Example 4
The labeled protein is compared with apoferritin-cisplatin compound and apoferritin-carboplatin compound prepared by depolymerization recombination method and apoferritin-cisplatin compound prepared by in-situ generation method, their forms remain intact, and their biochemical characteristics are basically unchanged. In FIG. 6, lane 1 is a marker protein; lane 2 is a apoferritin-carboplatin complex prepared by disaggregation recombination; lane 3 is a apoferritin-cisplatin complex prepared by disaggregation recombination; lane 4 is a apoferritin-cisplatin complex prepared by in situ generation. The illustration of the non-denaturing electrophoretic map shown in FIG. 6 shows: no matter which method is used for obtaining the product, the molecular weight of the product is near 440KDa after the product is wrapped by apoferritin cisplatin, or wrapped by carboplatin, or wrapped by a mixture of cisplatin and carboplatin, and the product is the same as ferritin, which shows that after the apoferritin is wrapped by platinum anti-cancer drugs, the protein shell is still intact, and the biochemical characteristics of the product are not changed basically.
Example 5
Cytotoxic activity experiments were tested using the MTT method: rat chromaffin neuroma cell PC-12 cell is selected as tumor model cell, and comparative experiment is carried out by adopting apoferritin obtained in example 1 and apoferritin-cisplatin compound prepared by in situ generation method in example 3.
PC12 cells were cultured using a 96-well plate at a cell density of 1X 104Per well, 100. mu.l of 1 and 2X 10 concentrations were added after 24 hours of incubation-6And (3) incubating the M apoferritin-cisplatin compound, apoferritin and cisplatin solution for 16 hours, removing the culture solution, washing the medium with a phosphoric acid buffer solution, adding a nutrient solution, and culturing the cells for 24 hours. To each well was added 10. mu.L of MTT solution ((5 mg. multidot.mL)-1) After 4 hours of reaction at 37 ℃ the solution was removed, 150. mu.L of DMSO was added to each well, and the plates were incubated at room temperature for 30 minutes and the absorbance at 490nm was measured using a microplate reader to calculate the viability of the cells in the different media.
FIG. 7 is a graph showing the results of an experiment in which apoferritin-cisplatin complex was inhibited against the rat chromaffin neuroma cell PC-12 cell line, in which: Af-CDDP is a apoferritin-cisplatin complex; af is apoferritin. The experimental results show that: the apoferritin-cisplatin compound prepared by the in-situ generation method has obvious inhibition effect on a common nerve tumor model cell (PC12), which shows that the cisplatin packaged in the apoferritin-cisplatin compound still has the effect of inhibiting the propagation of tumor cells.
The above-described embodiments are not intended to specifically limit the present invention.
Claims (7)
1. A apoferritin-platinum anticancer drug compound is characterized in that: the platinum anticancer drug is wrapped in apoferritin.
2. The apoferritin-platinum anticancer drug complex of claim 1 wherein: the platinum anticancer drug is cisplatin or carboplatin or a mixture of cisplatin and antiplatin.
3. The method for preparing apoferritin-platinum anticancer drug complex according to claim 1 or 2, wherein:
a) putting ferritin in acetic acid buffer solution with pH of 5.0-6.0 for dialysis, adding sodium hydrosulfite as a reducing agent and bipyridine as a chelating agent into dialysate, and replacing the dialysate every 8 hours until the dialysate is colorless and the ferritin is light yellow to obtain apoferritin;
b) the apoferritin and the platinum anticancer drugs are wrapped in the apoferritin by a depolymerization recombination method or an in-situ generation method to obtain the apoferritin-platinum anticancer drug compound.
4. The method for preparing apoferritin-platinum anticancer drug complex as claimed in claim 3, wherein: the platinum anti-cancer drug wrapped in the apoferritin by the depolymerization and recombination method is cisplatin or carboplatin, and the specific preparation method comprises the following steps: adding apoferritin into a saturated solution of a platinum anti-cancer drug, wherein the saturated solution of the platinum anti-cancer drug is a saturated solution of cisplatin or carboplatin, adjusting the pH value to 2.0-3.0 by using an acidic adjusting solution, slightly shaking for 30 minutes, adjusting the pH value to 6.0-9.0 by using an alkaline solution, and after incubation for two hours, completely dialyzing by using 0.1-0.2 mol/L sodium chloride solution to separate the apoferritin coated with the platinum anti-cancer drug from drug molecules which are not coated in the solution, thereby obtaining the apoferritin-platinum anti-cancer drug compound.
5. The method for preparing apoferritin-platinum anticancer drug complex as claimed in claim 4, wherein: diluting the apoferritin by using a sodium chloride aqueous solution, and then adding the diluted apoferritin into a saturated solution of a platinum anti-cancer drug, wherein the addition amount of the apoferritin in the saturated solution of cisplatin or carboplatin is as follows: the molar ratio of the apoferritin to the cisplatin in the saturated solution is 1: 1500-2500, and the molar ratioof the apoferritin to the carboplatin in the saturated solution is 1: 3000-4000; the acidic adjusting solution is hydrochloric acid adjusting solution, and the alkaline adjusting solution is sodium hydroxide solution.
6. The method for preparing apoferritin-platinum anticancer drug complex as claimed in claim 3, wherein: the in-situ generation method for generating the platinum anticancer drug in the apoferritin is a mixture containing cisplatin and anti-platinum, and the specific preparation method comprises the following steps: adding K with the concentration 500 times that of the protein into the apoferritin solution diluted by the sodium perchlorate aqueous solution2PtCl4Slightly shaking the solution for 30 minutes, and then adding equal volume of NH with the pH value of 9.0-10.5 into the solution3-(NH4)2SO4And (3) buffering the solution, reacting for 24 hours, and then thoroughly dialyzing the reaction product with a sodium perchlorate aqueous solution to separate apoferritin coated with the platinum anti-cancer drugs from reactants in the solution, thereby obtaining the apoferritin-platinum anti-cancer drug compound.
7. The method for preparing apoferritin-platinum anticancer drug complex as claimed in claim 6, wherein: and thoroughly dialyzing the reaction product by using 0.1-0.2 mol/L sodium perchlorate aqueous solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164339A (en) * | 2017-06-14 | 2017-09-15 | 中国科学院武汉病毒研究所 | A kind of method in protein nano cage interior packing allogenic material |
| CN107790185A (en) * | 2017-10-23 | 2018-03-13 | 南京大学 | Iron-sulfur cluster apoferritin compound and preparation method and application |
| CN109010847A (en) * | 2018-09-18 | 2018-12-18 | 昆明理工大学 | Apoferritin-benzothiazole compound compound and its preparation method and application |
| CN110237262A (en) * | 2018-03-07 | 2019-09-17 | 昆山新蕴达生物科技有限公司 | HFn contains the method and its product of platinum medicine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557822A (en) * | 2004-02-11 | 2004-12-29 | 昆明贵金属研究所 | Platinum complex having anti-tumor activity |
| CN1634946A (en) * | 2004-11-24 | 2005-07-06 | 昆明贵金属研究所 | Platinum complex for treating cancer and method for making same |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164339A (en) * | 2017-06-14 | 2017-09-15 | 中国科学院武汉病毒研究所 | A kind of method in protein nano cage interior packing allogenic material |
| CN107790185A (en) * | 2017-10-23 | 2018-03-13 | 南京大学 | Iron-sulfur cluster apoferritin compound and preparation method and application |
| CN107790185B (en) * | 2017-10-23 | 2020-05-05 | 南京大学 | Iron-sulfur cluster-apoferritin compound and preparation method and application thereof |
| CN110237262A (en) * | 2018-03-07 | 2019-09-17 | 昆山新蕴达生物科技有限公司 | HFn contains the method and its product of platinum medicine |
| CN110237262B (en) * | 2018-03-07 | 2021-06-08 | 昆山新蕴达生物科技有限公司 | HFn method for loading platinum medicine and its product |
| CN109010847A (en) * | 2018-09-18 | 2018-12-18 | 昆明理工大学 | Apoferritin-benzothiazole compound compound and its preparation method and application |
| CN109010847B (en) * | 2018-09-18 | 2021-07-16 | 昆明理工大学 | Apoferritin-benzothiazole compound and preparation method and application thereof |
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| Publication number | Publication date |
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| CN100418520C (en) | 2008-09-17 |
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